Document zopZgQV1wa7y6n2NNXgpXa9en

AR226-2860 Material Tested: , E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine HASKELL LABORATORY REPORT NO. 643-77 3 Haskell No. 11,336 Material Submitted By : Jprganic Chemicals Dept., '"'Jackson Laboratory Sample Ready for Testing: 6-23-77 --'' MUTAGENIC ACTIVITY OfM I IN THE SALMONELLA'/MICROSOME i tn\ Materials and Methods: Five histidine-requiring strains of Salmonella typhimumon were used in^the mutagen assays. Trains TA 1535 and TA 100 are used to detect base-pair substitution mutations, whereas strains iA 1537, TA 1538 and IA 98 are used to detect frame-shift mutations. (Ames, McCann and Yamasaki, Mutation Research 3J_ (1975) 347-364) The tests were performed in the pres nee and absence of a rat-liver homogenate activt ion system (S-9). |n t e absence of matabolic activation, 0.1 ml of a solution of the test compound and approximately 10 bacteriu were ucdec .c 2 ml of top agar (0.6% agar, 0.6NaCI, 0.05 mM L-histidine, 0.05 mM biotin). The solution was mixed nr.r) -ourec on ^ne surface of a Davis minimal agar plate. The metabolic activation system involved the addition of 0.5 ml oi S-9 mixiu - ^ to the chemical-tcp agar solution. The S-9 mix contains per ml : 0.3 ml of the 9,000 X g supernatant of homogenizoc ^rs. liver, 5 mM MgCl2, 33 mM KCI, 5 mM glucose-G-phosphate, 4 mM NADP and 100 .mM sodium phosphato (pH 7.4). Tnis mixture was adeed directly to the top agar immediately before It was poured over the minimal.agar plate. Prior to testing for mutagenicity, the compound is tested for toxicity. TA 1535 is routinely used to determine the general toxicity range for all strains. Appropriate controls were included for each strain. In the nonactivated system these controls consisted of ^^ negative, or solvent control, and positive controls. A second negative control (--S-9 control) is included in the a c * i v c . e u assay to measure any activity of the compound in the absence of the S-9 activator mixture. All plates were Incubated at 37C for 48 hours. Company Sanitized. Does not contain T SC A CBI 2- - Results: Tables I. and II. Summary: tested in Salmonella typhimuriuni strains TA 1535, TA 1537, TA 1538, TA vli end 7A 100 *" CCr.CT"" trationsijp to 10,000 pig Per petri plate. The compound was not mutagenic in the microbial assays either ,~ i 2r96" C& or absence of a liver microsomal system, i.e., it did not induce a significant increase over the spontaneous 'uTB" *cr. frequency. ' JFR:DFK:ms Date: Au gust IQ. IQ77 Approved by: David . Krahn Chief, Molecular Biology Secticr Company Sanitized. Does not contain TSCA C8I - TABLE I mutagenic activity STRAINS TA 1535. TA 1537, TA 53B. TA 98 AN / llA U iV otlC activ at ion 3 ComDOund Added _ h 2o 11,336 -S*9* 2AA yg/plate 10,000 2,000 4,000 H III 11 6,000 8,000 qi 11 10,000 FI 5 V 10 HI m o II Hlst|dlne+ Revertants Per Plate** TRT533 '-- TTT557---- YA 11338 TA 98 16 16 26 31 30 II 7 21 17 II 22 27 18 8 23 29 16 7 ?3 18 17 10 26 35 16 12 22 38 414 2Qil5 1927 620 H2 O 11,336 2AA ** 3 Dls+II lad water (solvent control) -(MD 3 2--Vninoanthracene (positive control) Test plate without S*9 and activators Average of two pIates TA 100 185 177 185 151 178 184 170 1966 Company Does no* contain T S C A CBJ TABLE II MUTAGENIC ACTIVITY OFIjflHflUrt ,N SALMONELLA TIPShfURIUM STRAINS TA 1535. TA 1537. TA I53& TA 98 AND TA 100 WITHOUT METABOLIC ACTIVATION Compound Added h 2o 11,336 MNNG 9AAc 2NF pg/plate 2,000 II 4,000 II 6,000 II 8,000 II 10,000 II 2 19 50 It 25 It TA l$35 23 26 27 31 22 26 1897 Histidine* Revertants Per Plate Ya' IM7 ! tA 1538 Ya 98 18 10 26 11 16 25 14 18 30 10 9 28 8 II 28 12 II 30 1213 1950 2348 H2 O MNNG 9AAc 2NF * c Distilled water (solvent control) -fmmn = N-MethyI -N'-nItro-N-nItrosoguanIdIne (positive control) 9-Amlnoser IdIne (positive control) 2-Nltrofluorene (positive control) * Average of two plates Ta I0 167 173 202 182 200 184 1846 (^rapap^ Samttteed. Poes noi eorsialimTSCA CBS